Isolation, Characterization, and Expression Analysis of NAC Transcription Factor from Andrographis paniculata (Burm. f.) Nees and Their Role in Andrographolide Production.

Andrographis paniculata (Burm. f.) Nees is an important medicinal plant known for its bioactive compound andrographolide. NAC transcription factors (NAM, ATAF1/2, and CUC2) play a crucial role in secondary metabolite production, stress responses, and plant development through hormonal signaling. In this study, a putative partial transcript of three NAC family genes (ApNAC83, ApNAC21 22 and ApNAC02) was used to isolate full length genes using RACE. Bioinformatics analyses such as protein structure prediction, cis-acting regulatory elements, and gene ontology analysis were performed. Based on in silico predictions, the diterpenoid profiling of the plant's leaves (five-week-old) and the real-time PCR-based expression analysis of isolated NAC genes under abscisic acid (ABA) treatment were performed. Additionally, the expression analysis of isolated NAC genes under MeJA treatment and transient expression in Nicotiana tabacum was performed. Full-length sequences of three members of the NAC transcription factor family, ApNAC83 (1102 bp), ApNAC21 22 (996 bp), and ApNAC02 (1011 bp), were isolated and subjected to the promoter and gene ontology analysis, which indicated their role in transcriptional regulation, DNA binding, ABA-activated signaling, and stress management. It was observed that ABA treatment leads to a higher accumulation of andrographolide and 14-deoxyandrographolide content, along with the upregulation of ApNAC02 (9.6-fold) and the downregulation of ApNAC83 and ApNAC21 22 in the leaves. With methyl jasmonate treatment, ApNAC21 22 expression decreased, while ApNAC02 increased (1.9-fold), with no significant change being observed in ApNAC83. The transient expression of the isolated NAC genes in a heterologous system (Nicotiana benthamiana) demonstrated their functional transcriptional activity, leading to the upregulation of the NtHMGR gene, which is related to the terpene pathway in tobacco. The expression analysis and heterologous expression of ApNAC21 22 and ApNAC02 indicated their role in andrographolide biosynthesis.

Decoding allelic diversity, transcript variants and transcriptional complexity of CENH3 gene in Brassica oleracea var. botrytis.

Histone proteins play a critical role in the primary organization of nucleosomes, which is the fundamental unit of chromatin. Among the five types of the histones, histone H3 has multiple variants, and the number differs among the species. Amongst histone H3 variants, centromeric histone H3 (CENH3) is crucial for centromere identification and proper chromosomal segregation during cell division. In the present study, we have identified 17 putative histone H3 genes of Brassica oleracea. Furthermore, we have done a detailed characterization of the CENH3 gene of B. oleracea. We showed that a single CENH3 gene exhibits allelic diversity with at least two alleles and alternative splicing pattern. Also, we have identified a CENH3 gene-specific co-dominant cleaved amplified polymorphic sequence marker SNP34(A/C) to distinguish CENH3 alleles and follow their expression in leaf and flower tissues. The gene structure analysis of the CENH3 gene revealed the conserved 5'-CAGCAG-3' sequence at the intron 3-exon 4 junction in B. oleracea, which serves as an alternative splicing site with one-codon (alanine) addition/deletion. However, this one-codon alternative splicing feature is not conserved in the CENH3 genes of wild allied Brassica species. Our finding suggests that transcriptional complexity and alternative splicing might play a key role in the transcriptional regulation and function of the CENH3 gene in B. oleracea. Altogether, data generated from the present study can serve as a primary information resource and can be used to engineer CENH3 gene towards developing haploid inducer lines in B. oleracea.

Development of genome-specific SSR markers for the identification of introgressed segments of Sinapis alba in the Brassica juncea background.

Sinapis alba L. (white mustard) is recognized for carrying host resistance against several biotic stresses including, Alternaria brassicae, which is responsible for blight disease in cultivated Brassica. However, another cultivated Brassica has a dearth for genetic resistance for these stresses due to its narrow genetic base. Therefore, we performed introgression of the genomic regions of S. alba into backcross progenies of B. juncea + S. alba somatic hybrids. These advanced generations with S. alba chromosomal segments are named B. juncea-S. alba introgression lines (ILs). In the present study, we developed the S. alba genome-specific microsatellites from the draft genome to track the S. alba genome introgressions and responsible regions for resistance to A. brassicae. For developing these SSR markers, the unique contigs of S. alba draft genome were identified through BLASTN with B. juncea, B. rapa, B. nigra, and B. oleracea reference genome assemblies, including mitochondrial and chloroplast genomes, and further used for marker development. Out of 403,423 contigs, we have identified 65,343 non-hit contigs of S. alba that yielded a total of 1231 genome-specific microsatellites, out of which 1107 were expected to produce a single allele upon amplification. Out of the total SSRs, 234 primer pairs were randomly picked from whole-genome and validated between B. juncea and S. alba genomes for their specificity. In the validation experiment, these markers gave a single amplicon into S. alba, while they did not amplify in B. juncea genome. Of these, 59 microsatellites were used to track S. alba introgressions in 80 BC2F3 lines. To the best of our knowledge, this is the first time that these two genetic resources are developed in the form of B. juncea-S. alba ILs and S. alba-specific markers. Therefore, both the resources unlock a new avenue of Brassica breeding for biotic and abiotic stresses along with quality traits. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03402-0.

Magnetopriming Actuates Nitric Oxide Synthesis to Regulate Phytohormones for Improving Germination of Soybean Seeds under Salt Stress.

In this study, the role of the signalling molecule nitric oxide (NO) in magnetopriming-mediated induction of salinity tolerance in soybean seeds is established. The cross-talk of NO with germination-related hormones gibberellic acid (GA), abscisic acid (ABA) and auxin (IAA) for their ability to reduce the Na+/K+ ratio in the seeds germinating under salinity is highlighted. Salt tolerance index was significantly high for seedlings emerging from magnetoprimed seeds and sodium nitroprusside (SNP, NO-donor) treatment. The NO and superoxide (O2•-) levels were also increased in both of these treatments under non-saline and saline conditions. NO generation through nitrate reductase (NR) and nitric oxide synthase-like (NOS-like) pathways indicated the major contribution of NO from the NR-catalysed reaction. The relative expression of genes involved in the NO biosynthetic pathways reiterated the indulgence of NR in NO in magnetoprimed seeds, as a 3.86-fold increase in expression was observed over unprimed seeds under salinity. A 23.26-fold increase in relative expression of NR genes by the NO donor (SNP) was observed under salinity, while the NR inhibitor (sodium tungstate, ST) caused maximum reduction in expression of NR genes as compared to other inhibitors [L-NAME (N(G)-nitro-L-arginine methyl ester; inhibitor of nitric oxide synthase-like enzyme) and DPI (diphenylene iodonium; NADPH oxidase inhibitor)]. The ratio of ABA/GA and IAA/GA decreased in magnetoprimed and NO donor-treated seeds, suggesting homeostasis amongst hormones during germination under salinity. The magnetoprimed seeds showed low Na+/K+ ratio in all treatments irrespective of NO inhibitors. Altogether, our results indicate that a balance of ABA, GA and IAA is maintained by the signalling molecule NO in magnetoprimed seeds which lowers the Na+/K+ ratio to offset the adverse effects of salinity in soybean seeds.

Plant growth-promoting rhizobacteria Shewanella putrefaciens and Cronobacter dublinensis enhance drought tolerance of pearl millet by modulating hormones and stress-responsive genes.

Drought is a major abiotic stress that affects crop productivity. Endophytic bacteria have been found to alleviate the adverse effects of drought on plants. In the present study, we evaluated the effects of two endophytic bacteria Shewanella putrefaciens strain MCL-1 and Cronobacter dublinensis strain MKS-1 on pearl millet (Pennisetum glaucum (L.) R. Br.) under drought stress conditions. Pearl millet plants were grown under three water levels: field capacity (FC), mild drought stress (MD), and severe drought stress (SD). The effects of inoculation on plant growth, physiological attributes, phytohormone content, and drought stress-responsive genes were assessed. The inoculation of pearl millet seeds with endophytes significantly improved shoot and root dry weight and root architecture of plants grown under FC and drought stress conditions. There was a significant increase in relative water content and proline accumulation in the inoculated plants. Among the phytohormones analyzed, the content of ABA and IAA was significantly higher in endophyte-treated plants under all moisture regimes than in uninoculated plants. C. dublinensis-inoculated plants had higher GA content than uninoculated plants under all moisture regimes. The expression level of genes involved in phytohormone biosynthesis (SbNCED, SbGA20oX, and SbYUC) and coding drought-responsive transcription factors (SbAP2, SbSNAC1 and PgDREB2A) was significantly higher under SD in endophyte-inoculated plants than in uninoculated plants. Thus, these endophytic bacteria presumably enhanced the tolerance of pearl millet to drought stress by modulating root growth, plant hormones, physiology and the expression of genes involved in drought tolerance.

Effect of Magnetopriming on Photosynthetic Performance of Plants.

Magnetopriming has emerged as a promising seed-priming method, improving seed vigor, plant performance and productivity under both normal and stressed conditions. Various recent reports have demonstrated that improved photosynthesis can lead to higher biomass accumulation and overall crop yield. The major focus of the present review is magnetopriming-based, improved growth parameters, which ultimately favor increased photosynthetic performance. The plants originating from magnetoprimed seeds showed increased plant height, leaf area, fresh weight, thick midrib and minor veins. Similarly, chlorophyll and carotenoid contents, efficiency of PSII, quantum yield of electron transport, stomatal conductance, and activities of carbonic anhydrase (CA), Rubisco and PEP-carboxylase enzymes are enhanced with magnetopriming of the seeds. In addition, a higher fluorescence yield at the J-I-P phase in polyphasic chlorophyll a fluorescence (OJIP) transient curves was observed in plants originating from magnetoprimed seeds. Here, we have presented an overview of available studies supporting the magnetopriming-based improvement of various parameters determining the photosynthetic performance of crop plants, which consequently increases crop yield. Additionally, we suggest the need for more in-depth molecular analysis in the future to shed light upon hidden regulatory mechanisms involved in magnetopriming-based, improved photosynthetic performance.

Magneto-priming promotes nitric oxide via nitric oxide synthase to ameliorate the UV-B stress during germination of soybean seedlings.

We have evaluated the contribution of nitric oxide (NO) in static magnetic field (SMF-200 mT for 1h) induced tolerance towards UV-B stress in soybean seedlings using various NO modulators like sodium nitroprusside (SNP), inhibitor of nitrate reductase (NR) sodium tungstate (ST), NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) and diphenylene iodonium (DPI) a NADPH oxidase inhibitor. The UV-B exposure significantly reduced germination, seedling growth together with activities of total amylase, NOS and NR in seedlings from un-primed seeds whereas SMF-primed seedlings showed significant enhancement in all these parameters along with higher level of NO/ROS. The supply of NO donor, SNP further improved all the seedlings parameters in un-primed and SMF-primed seeds after UV-B exposure. While ST, L-NAME and DPI significantly reduced the SMF-induced seedling performance after UV-B exposure. The gene expression study also showed significant up-regulation of α-amylase (GmAMY1, GmAMY2), nitric oxide synthase (GmNOS2) and nitrate reductase (GmNR2) encoding genes in UV-B exposed SMF-primed seedlings over un-primed seedlings. In particular, SNP+UV-B treatment enhanced the GmNOS2 expression in both unprimed (31.9-fold) and SMF-primed (93.2-fold) seedlings in comparison to their respective controls of CK+UV-B. In contrast, L-NAME+UV-B treatment reduced the SMF-induced GmNOS2 expression (4.8-fold) and NOS activity (76%). It confirmed that NO may be the key signaling molecule in SMF stimulated tolerance towards UV-B stress during early seedling growth and NOS may possibly be accountable for SMF-triggered NO production in soybean seedlings exposed to UV-B irradiations.

Analysis of Promoters of Arabidopsis thaliana Divergent Gene Pair SERAT3;2 and IDH-III Shows SERAT3;2 Promoter is Nested Within the IDH-III Promoter.

Intergenic regions of divergent gene pairs show bidirectional promoter activity but whether regulatory sequences for gene expression in opposite directions are shared is not established. In this study, promoters of divergently arranged gene pair At4g35640-At4g35650 (SERAT3;2-IDH-III) of Arabidopsis thaliana were analyzed to identify overlapping regulatory regions. Both genes showed the highest expression in flower buds and flowers. 5' RACE experiments extended the intergenic region from 161 bp shown in TAIR annotation to 512 bp. GUS analysis of transgenic A. thaliana plants carrying the 691 bp fragment (512 bp intergenic region plus 5' UTR of both the genes) linked to uidA gene revealed that SERAT3;2 promoter drives gene expression in the tapetum, whereas IDH-III promoter functions specifically in microspores/pollen. Serial 5' deletion of the 691 bp fragment showed SERAT3;2 promoter extends up to -355 position, whereas IDH-III promoter encompasses the 512 bp intergenic region. In transgenics, uidA transcript levels were lower than native SERAT3;2 and IDH-III transcripts indicating presence of additional cis regulatory elements beyond the 691 bp fragment. The present study demonstrated for the first time occurrence of a nested promoter in plants and identified a novel bidirectional promoter capable of driving gene expression in tapetum and microspores/pollen.